Paeonia lactiflora improves ovarian function and oocyte quality in aged female mice

Although ovarian aging is a key cause of decreased ovarian function and oocyte quality, it remains a problem in infertility treatment. Therefore, this study is aimed to investigate whether Paeonia lactiflora (PL), a herb improves ovarian function and oocyte quality using aged female mice. C57BL/6 female mice aged 8 months were treated orally every day with PL of 26.5 mg/kg (n=7) and 53 mg/kg (n=7) of body weight for 4 weeks using an oral zoned needle. The control group (n=7) was treated with normal saline. Ovaries and serum were collected for the H&E stain and the evaluation of reactive oxygen species (ROS) levels, respectively. In the second experiment, female mice were orally administered with PL (26.5 mg/kg: n=12, 53 mg/kg: n=12, control: n=12) and then superovulated with PMSG and hCG, and mated with male mice. Zygotes were retrieved and cultured for 4 days. Ovaries were provided for examination of expressions of genes associated with angiogenesis (VEGF and visfatin), anti-aging (Sirt1 and Sirt2), and follicular development (c-Kit, BMP-15, and GDF-9). PL significantly increased numbers of surviving follicles (primordial, primary, secondary, and antral), numbers of zygotes retrieved, embryo development rate, and ovarian expression of VEGF, visfatin, c-Kit, BMP-15, and GDF-9 at both doses. However, ovarian expression of Sirt1 and Sirt2 was increased at 53.0 mg/kg of PL. ROS levels were not affected by PL. These results suggest that PL may possess beneficial effects regarding ovarian function and oocyte quality, possibly by activation of ovarian angiogenesis and follicular development.

Paeonia lactiflora improves ovarian function and oocyte quality in aged female mice

Although ovarian aging is a key cause of decreased ovarian function and oocyte quality, it remains a problem in infertility treatment. Therefore, this study is aimed to investigate whether Paeonia lactiflora (PL), a herb improves ovarian function and oocyte quality using aged female mice. C57BL/6 female mice aged 8 months were treated orally every day with PL of 26.5 mg/kg (n=7) and 53 mg/kg (n=7) of body weight for 4 weeks using an oral zoned needle. The control group (n=7) was treated with normal saline. Ovaries and serum were collected for the H&E stain and the evaluation of reactive oxygen species (ROS) levels, respectively. In the second experiment, female mice were orally administered with PL (26.5 mg/kg: n=12, 53 mg/kg: n=12, control: n=12) and then superovulated with PMSG and hCG, and mated with male mice. Zygotes were retrieved and cultured for 4 days. Ovaries were provided for examination of expressions of genes associated with angiogenesis (VEGF and visfatin), anti-aging (Sirt1 and Sirt2), and follicular development (c-Kit, BMP-15, and GDF-9). PL significantly increased numbers of surviving follicles (primordial, primary, secondary, and antral), numbers of zygotes retrieved, embryo development rate, and ovarian expression of VEGF, visfatin, c-Kit, BMP-15, and GDF-9 at both doses. However, ovarian expression of Sirt1 and Sirt2 was increased at 53.0 mg/kg of PL. ROS levels were not affected by PL. These results suggest that PL may possess beneficial effects regarding ovarian function and oocyte quality, possibly by activation of ovarian angiogenesis and follicular development.(AU)

Simazine, a triazine herbicide, disrupts swine granulosa cell functions

The triazine herbicide simazine is a pesticide commonly detected in surface and ground waters, although banned in most European countries since 2004. Concerns for humans and animal health result from its potential endocrine disrupting action, that can lead to reproductive disorders. The present in vitro study was undertaken to study simazine effects on swine granulosa cell function, namely cell viability, proliferation, steroidogenesis and NO production. Moreover, the ability of this substance to interfere with the angiogenetic process, a crucial event in reproductive function, was taken into account. Our data document that simazine treatment, at 0.1 or 10 μM concentration levels, stimulates granulosa cell proliferation and viability and impairs steroidogenesis, increasing in particular progesterone production. In addition, the in vitro angiogenesis bioassay revealed a significant simazine stimulatory effect on immortalized porcine Aortic Endothelial Cell proliferation. Collectively, these results show that simazine can display disruptive effects on ovarian cell functional parameters, possibly resulting in reproductive dysfunction. This hypothesis is also supported by the observed pro-angiogenetic properties of this herbicide, as already suggested for different endocrine disruptors.

Simazine, a triazine herbicide, disrupts swine granulosa cell functions

The triazine herbicide simazine is a pesticide commonly detected in surface and ground waters, although banned in most European countries since 2004. Concerns for humans and animal health result from its potential endocrine disrupting action, that can lead to reproductive disorders. The present in vitro study was undertaken to study simazine effects on swine granulosa cell function, namely cell viability, proliferation, steroidogenesis and NO production. Moreover, the ability of this substance to interfere with the angiogenetic process, a crucial event in reproductive function, was taken into account. Our data document that simazine treatment, at 0.1 or 10 μM concentration levels, stimulates granulosa cell proliferation and viability and impairs steroidogenesis, increasing in particular progesterone production. In addition, the in vitro angiogenesis bioassay revealed a significant simazine stimulatory effect on immortalized porcine Aortic Endothelial Cell proliferation. Collectively, these results show that simazine can display disruptive effects on ovarian cell functional parameters, possibly resulting in reproductive dysfunction. This hypothesis is also supported by the observed pro-angiogenetic properties of this herbicide, as already suggested for different endocrine disruptors.(AU)

Transplante ovariano: Destaques na reprodução de primatas não humanos / Ovarian transplantation: highlight to non-human primates reproduction

Background: Ovarian transplantation in nonhuman primates (NHP) has been used as a strategy for the development of experimental models for biomedical research in the reproductive area. The prospects for application of this technique range from the restoration of female fertility to the conservation of endangered wild animals. However, studies with NHP were performed mostly focusing on the ovarian transplantation of cryopreserved tissue, in order to simulate the reality of human species, as well as aiming to obtain experimental models suitable for comparative studies. On the other hand, ovarian transplantation could be applied also in NHP species preservation.Review: According to the last census (2012-2014) of the International Union for Conservation of Nature (IUCN), currently, more than half of the 633 types of primates known around the world are in danger of disappearing forever due to the continued destruction of their natural habitat by human activities. Thus, there is an interest to expose the possible methods of ovarian preservation followed by transplantation that can be employed to promote the conservation of endangered NHP. Despite the positive results obtained with avascular autograft of fresh ovarian tissue in NHP, it is important to bring in mind the significant loss of follicles as a result of this procedure due to ischemia and reperfusion during the first days after grafting. This phenomenon leads to the production of reactive oxygen species (ROS) such as hydrogen peroxide, superoxide anion and hydroxyl radical, which are responsible by lipid peroxidation, cell membrane damage and subsequent follicular atresia. An alternative to counteract this oxidative stress consists in the application/administration of different sources of antioxidants previously or during grafting, in the grafted tissue or in the animal receiving the transplant. The most used compounds with a claimed radical scavenger activity are catalase, trolox and some vitamins.[...](AU)

Transplante ovariano: Destaques na reprodução de primatas não humanos / Ovarian transplantation: highlight to non-human primates reproduction

Background: Ovarian transplantation in nonhuman primates (NHP) has been used as a strategy for the development of experimental models for biomedical research in the reproductive area. The prospects for application of this technique range from the restoration of female fertility to the conservation of endangered wild animals. However, studies with NHP were performed mostly focusing on the ovarian transplantation of cryopreserved tissue, in order to simulate the reality of human species, as well as aiming to obtain experimental models suitable for comparative studies. On the other hand, ovarian transplantation could be applied also in NHP species preservation.Review: According to the last census (2012-2014) of the International Union for Conservation of Nature (IUCN), currently, more than half of the 633 types of primates known around the world are in danger of disappearing forever due to the continued destruction of their natural habitat by human activities. Thus, there is an interest to expose the possible methods of ovarian preservation followed by transplantation that can be employed to promote the conservation of endangered NHP. Despite the positive results obtained with avascular autograft of fresh ovarian tissue in NHP, it is important to bring in mind the significant loss of follicles as a result of this procedure due to ischemia and reperfusion during the first days after grafting. This phenomenon leads to the production of reactive oxygen species (ROS) such as hydrogen peroxide, superoxide anion and hydroxyl radical, which are responsible by lipid peroxidation, cell membrane damage and subsequent follicular atresia. An alternative to counteract this oxidative stress consists in the application/administration of different sources of antioxidants previously or during grafting, in the grafted tissue or in the animal receiving the transplant. The most used compounds with a claimed radical scavenger activity are catalase, trolox and some vitamins.[...]

Avanços na criopreservação de tecido ovariano de cabras e ovelhas / Advances in ovarian tissue cryopreservation in goats and sheep

A criopreservação de tecido ovariano, sem dúvidas, tem se tornado, uma prática não apenas suplementar as técnicas de reprodução assistida, mas também fundamental para a preservação da fertilidade da fêmea. Isso tem sido observado, especialmente na espécie humana, cuja espécie em uma década de trabalho, já se soma aproximadamente trinta nascimentos de bebês saudáveis oriundos de ovário criopreservado. No entanto, todo o avanço que hoje é estabelecido, muito se deve aos trabalhos pioneiros realizados em animais de animais de produção como os ovinos. Relatos de sucesso após criopreservação e transplante de tecido ovariano no início dos anos 90 impulsionaram importantes equipes a investir esforços e dedicação nessa área, voltados para a reprodução humana, e embora, o sucesso seja evidente, os avanços da criopreservação de tecido ovariano tanto na espécie ovina, como na espécie caprina são ainda bastante limitados. Portanto, o objetivo desse trabalho, é apresentar uma retrospectiva dos principais resultados obtidos com a criopreservação de tecido ovariano nessas duas espécies.(AU)

Cryopreservation of ovarian tissue, no doubt, has been considerated not only as an additional practice to assisted reproductive techniques, but it is fundamental for female fertility preservation. This have been related particularly in humans, whose specie in a decade, it was reported approximately thirty healthy babies born after procedures of cryopreservation and tranplant of ovarian tissue. However, the currently successful is due to hard efforts done in animals farm such as sheep. Reports of success after cryopreservation and transplantation of ovarian tissue in the early 90 stimulated several researchers to invest efforts and dedication in that research field, regarding human reproduction, and although success is evident, advances in cryopreservation of ovarian tissue in sheep and goats are still quite limited. Therefore, the aim of this work is to present a retrospective of the main results obtained with the cryopreservation of ovarian tissue in these two species.(AU)

UTILIZAÇÃO DO PROCESSO BIOTECNOLÓGICO DO OVÁRIO ARTIFICIAL PARA AVALIAR A TOXICIDADE DO VITONOLIDO D E DA QUINOXALINA SOBRE OS FOLÍCULOS PRÉ-ANTRAIS CAPRINOS

Embora o número de novos casos de câncer venha aumentando anualmente, o número de sobreviventes também vem crescendo ao longo dos anos, em função do diagnostico precoce e de tratamentos quimioterápicos mais efetivos. Porém, dentre os grandes efeitos indesejáveis da quimioterapia, destaca-se a resistência a multi-drogas (RMD) e a falha ovariana que leva à infertilidade. Portanto, a presente estudo teve dois principais objetivos: 1) imunolocalizar membros da família de proteínas conhecidas como Transportadores ABC (ABCB1, ABCC2 e ABCG2) no tecido ovariano caprino (Fase 1) e 2) avaliar a toxicidade de dois novos compostos com potencial anticâncer, ou seja, o Vitanolido D - VD (Fase 2) e a Quinoxalina - QX (Fase 3) sobre os folículos pré-antrais (primordiais, primários e secundários) cultivados in vitro no interior do tecido ovariano. Na Fase 1, ovários de cabras foram processados e analisados por imuno-histoquímica, qPCR e Western Blottingg para a identificação do ABCB1, ABCC2 e ABCG2. Nas Fases 2 e 3, fragmentos do córtex ovariano foram cultivadas in vitro por 2 e 6 dias em -MEM+ ou -MEM+ adicionado de paclitaxel (PTX controle negativo) ou em diferentes concentrações de VD (1.5, 3.0 e 6.0 M) ou QX (1.5, 3.0 e 6.0 M), respectivamente. Nessas duas fases os folículos foram analisados antes (controle) e após o cultivo na presença dos compostos, considerando-se morfologia e ativação folicular; proliferação (Ki67) e apoptose (TUNEL) das células granulosa, a expressão proteica do ABCB1 e gênica dos fatores reguladores do ciclo celular ciclinas (A - CCNA, B1 - CCNB1, D1 - CCND1 e E1 - CCNE1) e quinases dependentes de ciclina (1 - CDK1; 2 - CDK2, 4 - CDK4 e 6 - CDK6)]. Os resultados mostraram que os três transportadores ABCs foram positivamente imunomarcadas em todas as categorias foliculares estudadas. No tocante aos compostos, a toxicidade do VD6.0 foi similar ao PTX e superior (P <0,05) às demais concentrações (1.5 e 3.0 M), após 2 e 6 dias. A QX também apresentou alta toxicidade aos folículos pré-antrais, especialmente na mais alta concentração (6.0 M), causando um bournout folicular e apoptose, superiores às concentrações de 1.5 e 3.0 M. Ao contrário do VD, a QX inibiu a expressão do ABCB1 nos folículos pré-antrais. Esse trabalho mostrou pela primeira vez que três membros da família dos ABC transportadores foram identificados no ovário caprino. Apesar dos valiosos resultados obtidos nesse estudo, os dois compostos, isto é, o VD e a QX, necessitam ser melhor investigados, sob o ponto de vista da função ovariana, visando auxiliar na sua utilização como agentes quimioterápicos com menor risco para a fertilidade feminina.

Although the number of new cases of cancer increases annually, the number of survivors has also increased over the years, due to the early diagnosis and more effective chemotherapy treatments. However, among the great undesirable effects of chemotherapy, the multidrug resistance (MDR) and the ovarian failure that lead to infertility. Therefore, the present thesis had two main objectives: 1) to immunolocalize members of the family of proteins known as ABC transporters (ABCB1, ABCC2 and ABCG2), responsible for the phenomenon of RMD in goat ovarian tissue (Phase 1) and 2) to evaluate the toxicity of two new compounds with anticancer potential, the Vitanolide D - VD (Phase 2) and Quinoxaline - QX (Phase 3) on the preantral follicles (primordial, primary and secondary) grown in vitro inside the ovarian tissue. In Phase 1, goat ovaries were processed and analyzed by immunohistochemistry, qPCR and Western Blotting for identification of ABCB1, ABCC2 and ABCG2. In Phases 2 and 3, fragments of the ovarian cortex were cultured in vitro for 2 or 6 days in -MEM+ or -MEM+ supplemented with paclitaxel (PTX - negative control) or at different RV concentrations (1.5, 3.0 and 6.0 M) or QX (1.5, 3.0 and 6.0 M), respectively. In these two phases the follicles were analyzed before (control) and after the culture in the presence of the compounds, considering morphology and follicular activation; proliferation (Ki67) and apoptosis (TUNEL) of the granulosa cells, the protein expression of ABCB1 and the gene of cyclin - cyclin regulatory factors (A - CCNA, B1 - CCNB1, D1 - CCND1 and E1 - CCNE1) and cyclin - dependent kinases 1-CDK1; 2-CDK2, 4-CDK4 and 6-CDK6)]. The results showed that the three ABCs transporters were positively immunolabelled in all follicular categories studied. Regarding the compounds, the toxicity of VD6.0 was similar to PTX and higher (P < 0.05) at the other concentrations (1.5 and 3.0 M) after 2 and 6 days. QX also showed high toxicity to the preantral follicles, especially in the highest concentration (6.0 M), causing a follicular burnout and apoptosis, higher than the concentrations of 1.5 and 3.0 M. Unlike VD, QX inhibited the expression of ABCB1 in preantral follicles. This work showed for the first time that three members of the family of ABC transporters were identified in ovary goat. Despite the valuable results obtained in this study, the two compounds, i.e., RV and QX, need to be better investigated, from the point of view of ovarian function, aiming to aid in their use as chemotherapeutic agents with lower risk for female fertility.

Avanços na criopreservação de tecido ovariano de cabras e ovelhas / Advances in ovarian tissue cryopreservation in goats and sheep

A criopreservação de tecido ovariano, sem dúvidas, tem se tornado, uma prática não apenas suplementar as técnicas de reprodução assistida, mas também fundamental para a preservação da fertilidade da fêmea. Isso tem sido observado, especialmente na espécie humana, cuja espécie em uma década de trabalho, já se soma aproximadamente trinta nascimentos de bebês saudáveis oriundos de ovário criopreservado. No entanto, todo o avanço que hoje é estabelecido, muito se deve aos trabalhos pioneiros realizados em animais de animais de produção como os ovinos. Relatos de sucesso após criopreservação e transplante de tecido ovariano no início dos anos 90 impulsionaram importantes equipes a investir esforços e dedicação nessa área, voltados para a reprodução humana, e embora, o sucesso seja evidente, os avanços da criopreservação de tecido ovariano tanto na espécie ovina, como na espécie caprina são ainda bastante limitados. Portanto, o objetivo desse trabalho, é apresentar uma retrospectiva dos principais resultados obtidos com a criopreservação de tecido ovariano nessas duas espécies.

Cryopreservation of ovarian tissue, no doubt, has been considerated not only as an additional practice to assisted reproductive techniques, but it is fundamental for female fertility preservation. This have been related particularly in humans, whose specie in a decade, it was reported approximately thirty healthy babies born after procedures of cryopreservation and tranplant of ovarian tissue. However, the currently successful is due to hardefforts done in animals farm such as sheep. Reports of success after cryopreservation and transplantation of ovarian tissue in the early 90 stimulated several researchers to invest efforts and dedication in that research field, regarding human reproduction, and although success is evident, advances in cryopreservation of ovarian tissue in sheep and goats are still quite limited. Therefore, the aim of this work is to present a retrospective of the main results obtained with the cryopreservation of ovarian tissue in these two species.